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Background Transgenic (Tg) mice are widely used in biomedical research, and they are typically generated by injecting transgenic DNA cassettes into pronuclei of one-cell stage zygotes. Such animals often show unreliable expression of the transgenic DNA, one of the major reasons for which is random insertion of the transgenes. We previously developed a method called “pronuclear injection-based targeted transgenesis” (PITT), in which DNA constructs are directed to insert at pre-designated genomic loci. PITT was achieved by pre-installing so called landing pad sequences (such as heterotypic LoxP sites or attP sites) to create seed mice and then injecting Cre recombinase or PhiC31 integrase mRNAs along with a compatible donor plasmid into zygotes derived from the seed mice. PITT and its subsequent version, improved PITT ( i -PITT), overcome disadvantages of conventional Tg mice such as lack of consistent and reliable expression of the cassettes among different Tg mouse lines, and the PITT approach is superior in terms of cost and labor. One of the limitations of PITT, particularly using Cre -mRNA, is that the approach cannot be used for insertion of conditional expression cassettes using Cre- LoxP site-specific recombination. This is because the LoxP sites in the donor plasmids intended for achieving conditional expression of the transgene will interfere with the PITT recombination reaction with LoxP sites in the landing pad. Results To enable the i -PITT method to insert a conditional expression cassette, we modified the approach by simultaneously using PhiC31o and FLPo mRNAs. We demonstrate the strategy by creating a model containing a conditional expression cassette at the Rosa26 locus with an efficiency of 13.7%. We also demonstrate that inclusion of FLPo mRNA excludes the insertion of vector backbones in the founder mice. Conclusions Simultaneous use of PhiC31 and FLP in i -PITT approach allows insertion of donor plasmids containing Cre- loxP -based conditional expression cassettes.

Male infertility affects approximately 7% of the male population, and about 15% of these cases are predicted to have a genetic etiology. One gene implicated in autosomal dominant male infertility, SYCP2, encodes a protein critical for the synapsis of homologous chromosomes during meiosis I, resulting in impaired spermatogenesis. However, the clinical validity of the gene–disease pair was previously categorized as on the border of limited and moderate due to few reported cases. This study investigates the genetic cause of infertility for three unrelated Chinese patients with oligoasthenozoospermia. Whole exome sequencing (WES) and subsequent Sanger sequencing revealed novel heterozygous loss-of-function (LOF) variants in SYCP2 (c.89dup, c.946_947del, and c.4378_4379del). These cases, combined with the previously reported cases, provide strong genetic evidence supporting an autosomal dominant inheritance pattern. The experimental evidence also demonstrates a critical role for SYCP2 in spermatogenesis. Collectively, this updated assessment of the genetic and experimental evidence upgrades the gene–disease association strength of SYCP2 and autosomal dominant male infertility from on the border of limited and moderate to strong. The reclassification improves SYCP2 variant interpretation and qualifies it for the inclusion on diagnostic male infertility gene panels and prioritization in whole exome or genome studies for related phenotypes. These findings therefore improve the clinical interpretation of SYCP2 LOF variants.

Human genetics research employs the two opposing approaches of forward and reverse genetics. While forward genetics identifies and links a mutation to an observed disease etiology, reverse genetics induces mutations in model organisms to study their role in disease. In most cases, causality for mutations identified by forward genetics is confirmed by reverse genetics through the development of genetically engineered animal models and an assessment of whether the model can recapitulate the disease. While many technological advances have helped improve these approaches, some gaps still remain. CRISPR/Cas (clustered regularly interspaced short palindromic repeats/CRISPR-associated), which has emerged as a revolutionary genetic engineering tool, holds great promise for closing such gaps. By combining the benefits of forward and reverse genetics, it has dramatically expedited human genetics research. We provide a perspective on the power of CRISPR-based forward and reverse genetics tools in human genetics and discuss its applications using some disease examples.

Background: Vanishing twin syndrome (VTS) represents a complex and under-recognized phenomenon in multifetal pregnancies, associated with both clinical uncertainty and significant psychosocial impact. Despite its frequency, gaps remain in diagnostic clarity, international guidelines, and communication strategies with patients and families. Materials and Methods: This hybrid review integrates narrative and systematic elements to assess the diagnostic, clinical, and psychosocial gaps in VTS. A systematic literature search was conducted across Medline/PubMed, CINAHL, PsycINFO, EBM Reviews, and Scopus using terms such as “vanishing twin syndrome,” “patient-provider communicat*,” and “bereave* care.” Sources included systematic reviews, randomized controlled trials, cohort studies, and qualitative studies. Exclusion criteria were outdated publications (>10 years old). Results: Evidence revealed multiple domains of concern. Clinical risks and diagnostics remain poorly defined, with inconsistent recognition of maternal and neonatal complications. Psychosocial impacts were prominent, encompassing grief, identity disruption, and unmet support needs. Patient–provider communication was frequently inadequate, with insufficient training and lack of standardized language. International guidelines varied widely in scope, with only a few of them providing clear recommendations for bereavement care in multifetal loss contexts. Discussion: Emerging discourse highlights the limitations of the traditional fission model and alternative conceptual frameworks, such as Herranz’s model, for understanding VTS. These theoretical differences underscore the need for precise terminology and consistent diagnostic practices. Clinical implications extend to prenatal screening, obstetric management, and the integration of psychosocial support. Patient-centered communication and structured support initiatives (e.g., the Butterfly Project) demonstrate the potential to bridge communication gaps and improve care experiences. Conclusions: VTS requires recognition as both a medical and psychosocial condition. Improved clinical definitions, harmonized international guidelines, and emphasis on empathetic communication are essential to address the current gaps. Integrating these elements into practice may enhance patient outcomes and provide families with validation and support following multifetal loss.

Recent advances in molecular cytogenetics highlight the importance of noncoding structural variation in human disease. Genomic rearrangements can disrupt chromatin architecture, leading to long-range alterations in gene expression. With increasing ability to assess distal gene dysregulation comes new challenges in clinical interpretation of rearrangements. While haplotyping methods to determine compound heterozygosity in a single gene with two pathogenic variants are established, such methods are insufficient for phasing larger distances between a pathogenic variant and a genomic rearrangement breakpoint. Herein, we present an inexpensive and efficient proximity ligation-based method called 3C-PCR for phasing chromosomal rearrangement breakpoints with distal allelic variants. 3C-PCR uses canonical chromosome conformation capture (3C) libraries for targeted distal phasing by implementing a novel nested PCR strategy with primers anchored across the rearrangement breakpoints and subsequent Sanger sequencing. As a proof of concept, 3C-PCR was used to phase a highly variable region 1.3 Mb upstream of a chromosomal rearrangement breakpoint in a balanced translocation. We found that the nested PCR approach amplified the derivative chromosome substrate exclusively and identified the same haplotype by Sanger sequencing reliably. Given its efficacy and versatility, 3C-PCR is ideal for use in phasing chromosomal rearrangement breakpoints with allelic variants located at a genomic distance over a megabase.

Purpose of Review Infertility affects 10–15% of couples, making it one of the most frequent health disorders for individuals of reproductive age. The state of childlessness and efforts to restore fertility cause substantial emotional, social, and financial stress on couples. Male factors contribute to about half of all infertility cases, and yet are understudied relative to female factors. The result is that the majority of men with infertility lack specific causal diagnoses, which serves as a missed opportunity to inform therapies for these couples. Recent Findings In this review, we describe current standards for diagnosing male infertility and the various interventions offered to men in response to differential diagnoses. We then discuss recent advances in the field of genetics to identify novel etiologies for formerly unexplained infertility. Summary With a specific genetic diagnosis, male factors can be addressed with appropriate reproductive counseling and with potential access to assisted reproductive technologies to improve chances of a healthy pregnancy.

With rapidly declining costs, whole genome sequencing is becoming feasible for widespread use. Although cost-effectiveness is driving increased use of the technology, comprehensive recommendations on how to handle ethical dilemmas have yet to reach a consensus. In this article, Sam shares her experience of undergoing whole genome sequencing. Despite the deeply private nature of the test, the results do not solely belong to Sam; her identical twin sister, Arielle, shares virtually the same genome and received results without a formal consent process. This article explores their parallel experiences as a way of highlighting the controversial ethics of a private test with familial implications.

Analysis of chromosomal rearrangements has been highly successful in identifying genes involved in many congenital abnormalities including hearing loss. Herein, we report a subject, designated DGAP242, with congenital hearing loss (HL) and a de novo balanced translocation 46,XX,t(1;5)(q32;q15)dn. Using multiple next-generation sequencing techniques, we obtained high resolution of the breakpoints. This revealed disruption of the orphan receptor ESRRG on chromosome 1, which is differentially expressed in inner ear hair cells and has previously been implicated in HL, and disruption of KIAA0825 on chromosome 5. Given the translocation breakpoints and supporting literature, disruption of ESRRG is the most likely cause for DGAP242's phenotype and implicates ESRRG in a monogenic form of congenital HL, although a putative contributory role for KIAA0825 in the subject's disorder cannot be excluded.

Infertility is one of the most common disorders for men of reproductive age. To identify novel genetic etiologies, we studied a male with severe oligozoospermia and 46,XY,t(20;22)(q13.3;q11.2). We identified exclusive overexpression of SYCP2 from the der(20) allele that is hypothesized to result from enhancer adoption. Modeling the dysregulation in budding yeast resulted in disruption of the synaptonemal complex, a common cause of defective spermatogenesis in mammals. Exome sequencing of infertile males revealed three novel heterozygous SYCP2 frameshift variants in additional subjects with cryptozoospermia and azoospermia. This study provides the first evidence of SYCP2-mediated male infertility in humans. Footnotes * https://github.com/mnnshreya/Morton-DGAP230